Central to the vectorial transport function of renal epithelium is the polarized distribution of surface membrane proteins in renal tubule cells. The spectrin-based membrane skeleton organizes mosaics of membrane proteins and lipids, and tethers them to cytoskeletal filament systems and motor proteins (e.g. dynein/dynactin). A working hypothesis that has emerged from recent data is that a novel Golgi-associated forms of spectrin and ankyrin, acting along the secretory and endocytic pathways, influences the efficiency of assembly for certain membrane proteins by forming a spectrin-ankyrin-adapter protein tethering system (SAATS). One such protein is alpha-Na,K-ATPase, which requires spectrin and ankyrin-binding early in the secretory pathway for its efficient and polarized delivery to the plasma membrane. Multiple recently discovered isoforms of spectrin and ankyrin also exist in the renal cell. The overall goal of the proposed studies is thus to understand the specific steps in the secretory pathway on which spectrin and or ankyrin act; understand which membrane proteins require spectrin or ankyrin for their efficient delivery to the membrane; and understand the aberrations in this process that typify cellular ATP depletion, a model of cellular ischemia. Research will focus on the role of beta-Ia nd beta-III spectrins and the role that two renal ankyrins (AnkG119 & AnkG190) play in the secretory pathway, as they interact with Na,K-ATPase. Three specific aims are proposed. Time-lapse video microscopy and FRET analysis using eGFP tagged spectrin peptides expressed in MDCK, COS, and LLC-PK1 cells will be used to follow the in vivo movement and associations of spectrin and ankyrin with various organelle and cargo proteins. Specific interactions of ankyrin with alpha-Na,K-ATPase will be studied to determine if ankyrin acts like a chaperone or a chauffeur in the process of membrane biogenesis. The consequences of ATP depletion in model cell systems on the activities of spectrin or ankyrin will be examined. Collectively, these studies will answer important questions that have followed from the discovery of a heretofore-unanticipated role for the spectrin-ankyrin cytoskeleton in the secretory pathway, and will provide insights into the role of certain novel protein-protein interactions, interactions that may afford novel targets for therapeutic intervention.